A regulatory region of the human transferrin receptor gene promoter was found to be required for increased expression in response to serum or growth factors. This region contains two elements that appear to cooperate for full responsiveness. We found that sodium orthovanadate treatment of cells significantly activated expression of promoter constructs containing these elements. 12-0-Tetradecanoylphorbol-13-acetate alone induced a twofold increase in expression but acted synergistically with vanadate to generate a highly elevated level of expression. Dibutyryl cyclic AMP alone had no effect on expression, but when added together with vanadate and 12-0-tetradecanoylphorbol-13-acetate, led to superinduction of the promoter construct. Induction of expression by these reagents was delayed several hours, and the kinetics were identical to those observed for serum induction.Transferrin receptors (TR) mediate uptake of iron into cells by binding and endocytosis of transferrin. Expression of TR is coupled to cell proliferation, and blocking of receptor function prevents cells from entering the S phase. This is most likely the result of an increased need for iron during the DNA-synthetic phase of the cell cycle. Rapidly proliferating cells have elevated levels of TR compared with quiescent, noncycling cells (13,22). When quiescent cells are activated by growth factors or mitogens, the level of cell surface TR increases. Increased expression of TR in mitogen-activated cells involves increased transcription of the TR-encoding gene (21). This is a delayed response that occurs several hours after mitogen stimulation and reaches a maximum just prior to entry into the S phase.Previously we have characterized the promoter region of the TR-encoding gene and analyzed the DNA-protein interactions within this region. We found two elements that are protected in DNase I footprinting experiments (3,26,28). These protein recognition sites are indicated by the boxes labelled A and B in Fig. 1A. Element A is GC rich and contains a consensus binding sequence for transcription factor Spl. However, we have demonstrated that multiple factors can interact within this region (26). Element B contains a sequence that is similar to the sequences recognized by both the Apl and cyclic AMP (cAMP) response element-binding protein (CREB) families of transcription factors. Microinjection of oligonucleotides that span both elements A and B was able to block serum induction of DNA synthesis (27). However, oligonucleotides for either site alone had no effect. These results suggest that the factors that recognize these sequences in the TR promoter have cooperative effects that are critical for the signalling pathways involved in transition to the S phase.